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Background: Cardiovascular disease (CVD) is a major cause of mortality in the United Kingdom. Epidemiologic studies suggest that consumption of tomato-based foods may lower CVD risk. Such potential benefits have been ascribed in part to high concentrations of lycopene in the tomatoes. However, these findings have not yet been validated by comprehensive intervention trials. Objective: The aim of this study was to conduct a single-blind, randomized controlled intervention trial with healthy middle-aged volunteers to assess whether the consumption of tomato-based foods affects recognized biomarkers of CVD risk. Design: After a 4-wk run-in period with a low-tomato diet, 225 volunteers (94 men and 131 women) aged 40‐65 y were randomly assigned into 1 of 3 dietary intervention groups and asked to consume a control diet (low in tomato-based foods), a high-tomato-based diet, or a control diet supplemented with lycopene capsules (10 mg/d) for 12 wk. Blood samples were collected at baseline, at 6 wk, and after the intervention and were analyzed for carotenoid and lipid profiles and inflammatory markers. Blood pressure, weight, and arterial stiffness were also measured. Dietary intake was also determined during the intervention. Results: None of the systemic markers (inflammatory markers, markers of insulin resistance and sensitivity) changed significantly after the dietary intervention. Moreover, lipid concentrations and arterial stiffness were also unaffected by the interventions. Conclusion: These data indicate that a relatively high daily consumption of tomato-based products (equivalent to 32‐50 mg lycopene/d) or lycopene supplements (10 mg/d) is ineffective at reducing conventional CVD risk markers in moderately overweight, healthy, middle-aged individuals. This trial was registered at isrctn.org as ISRCTN34203810. Am J Clin Nutr 2012;95:1013‐22.

Despite fruit, vegetables and many processed products counting towards achieving the recommended five-a-day strategy, it is inevitable that produce choice will affect the benefits delivered. Fruits locally produced and commonly consumed in Scotland were compared for their phenolic acid content and form. The phenolic acid composition was highly variable, but the locally produced fruits were significantly ( p – 4.89 g/kg compared to 0.06 – 0.22 g/kg). The majority of the phenolic acids were conjugated to other plant components, suggesting that any health benefits derived from these compounds are likely to be after they are released/metabolised by the colonic microbiota. Although the potential protective effects of the individual compounds will not be ascertained until the exact role of these compounds in disease prevention has been clarified, it is clear that the total amount of phenolic acids in the diet will vary enormously depending on the types of fruits consumed.

Evidence from a wide range of sources suggests that individuals taking aspirin and related non-steroidal anti-inflammatory drugs have reduced risk of large bowel cancer. Work in animals supports cancer reduction with aspirin, but no long-term randomised clinical trials exist in human beings, and randomisation would be ethically unacceptable because vascular protection would have to be denied to a proportion of the participants. However, opportunistic trials of aspirin, designed to test vascular protection, provide some evidence of a reduction in cancer, but only after at least 10 years. We summarise evidence for the potential benefit of aspirin and natural salicylates in cancer prevention. Possible mechanisms of action and directions for further work are discussed, and implications for clinical practice are considered.

The dietary phenolic compound, salicylic acid, decreases oxidative stress and pro-inflammatory and potentially neo-plastic prostaglandins with a concomitant increase in glutathione peroxidase activity. Salicylic acid, a dietary plant-based phenolic compound and also the main metabolite of aspirin, may contribute to the colon protective effects of plant-based diets. Oxidative stress is a characteristic of pre-cancerous and cancerous colon and inflammatory bowel diseases (IBD) that increase colon cancer risk. The mechanism(s) whereby salicylic acid modulates potentially pro-cancerous activity associated with oxidative stress is further investigated here using a proteomic approach. A rat model of oxidative stress was supplemented with salicylic acid (1 mg/kg diet, mean plasma levels 310+/-32 micromol/l). Soluble colon protein extracts were subjected to 2D PAGE. Salicylic acid modulated proteins, identified using MALDI-TOF and LC/MS/MS, are involved in protein folding, transport, redox, energy metabolism and cytoskeletal regulation. A partial least squares (PLS) analysis approach was used to assist biological interpretation of the altered protein profiles via their associations between previously published biochemical measurements of oxidative stress, prostaglandin levels and increased glutathione peroxidase activity. Early detection of altered homeostasis in colon may assist in identifying pre-pathological features preceding colon tumorigenesis and contribute to an understanding of epidemiological evidence supporting a protective effect of plant-based diets.

Oxidative stress is a characteristic of cancerous colon tissue and inflammatory bowel diseases that increase colon cancer risk. Epidemiological evidence supports a protective effect of plant-derived compounds. Aspirin is also protective against colon cancer. The mechanism of action is unclear although salicylic acid, the main metabolite of aspirin, has been shown to decrease the synthesis of pro-inflammatory and potentially neo-plastic prostaglandins. Salicylic acid is found in significant quantities in a plant-based diet. However, in plants salicylic acid is also reported to modulate the expression of numerous enzymes with antioxidant activity. The aim of this study was to assess whether salicylic acid can modulate pro-cancerous biological pathways in the colon. Oxidative stress, prostaglandins and cytosolic glutathione peroxidase (cyGPX) were analysed in proximal, transverse and distal colon from a rat model of diet-induced oxidative stress. Elevated plasma pyruvate kinase activity (1293+/-206 U/ml) and increased indices of lipid peroxidation in colon (proximal 6.4+/-0.84 nM MDA/mg protein; transverse 6.9+/-0.97 nM MDA/mg protein; distal 5.2+/-0.62 nM MDA/mg protein) from rats fed a Vitamin E deficient diet were significantly decreased on supplementation with salicylic acid (plasma pyruvate 546+/-43 U/ml; salicylic acid proximal 3.6+/-0.39 nM MDA/mg protein; transverse 4.5+/-0.61 nM MDA/mg protein; distal 4.4+/-0.27 nM MDA/mg protein). Reductions in oxidative stress and prostaglandin production on supplementation with salicylic acid were associated with an elevation in glutathione peroxidase activity (Vitamin E deficient proximal 0.056+/-0.013 U/mg protein; transverse 0.073+/-0.008 U/mg protein; distal 0.088+/-0.010 U/mg protein; Vitamin E deficient with salicylic acid proximal 0.17+/-0.01 U/mg protein; transverse 0.23+/-0.016 U/mg protein; distal 0.16+/-0.020 U/mg protein). Gpx1 and Gpx2 gene transcripts were not elevated in association with increased activity of the soluble glutathione peroxidase activity. Glutathione peroxidases are key antioxidant enzymes, catalysing the decomposition of potentially toxic lipid peroxides. Gpx activity and regulation of Gpx gene transcription has been shown previously to be complex with activity not necessarily mirrored by a corresponding elevation in gene transcription. By supplementing the diet of Vitamin E deficient rats with salicylic acid (1 g/kg diet), this study assessed effects of salicylic acid on cytosolic glutathione peroxidase activity in the colon. The ability of salicylic acid to modulate antioxidant enzymes in colon tissue may be an important mechanism in inhibiting colon cancer development.

The flavonol myricetin, reacts with oxygen-centred galvinoxyl radicals 28 times faster than d-α-tocopherol (vitamin E), the main lipid-soluble antioxidant in biological membranes. Moreover, each myricetin molecule reduces twice as many such radicals as vitamin E. However, myricetin fails to protect vitamin E-deficient microsomes from lipid peroxidation as assessed by the formation of thiobarbituric acid reactive substances (TBARS). Novel and potentially therapeutic antioxidants have been prepared that combine the radical-scavenging ability of a myricetin-like head group with a lipophilic chain similar to that of vitamin E. C 6 –C 12 alkyl chains are attached to the A-ring of either a 3,3 ′ ,4 ′ ,5 ′ -tetrahydroxyflavone or a 3,2 ′ ,4 ′ ,5 ′ -tetrahydroxyflavone head group to give lipophilic flavonoids ( C log P =4 to 10) that markedly inhibit iron-ADP catalysed oxidation of microsomal preparations. Orientation of the head group as well as total lipophilicity are important determinants of antioxidant efficacy. MM2 models indicate that our best straight chain 7-alkylflavonoids embed to the same depth in the membrane as vitamin E. The flavonoid head groups are prepared by aldol condensation followed by Algar–Flynn–Oyamada (AFO) oxidation or by Baker–Venkataraman rearrangement. The alkyl tails are introduced by Suzuki or Negishi palladium-catalysed cross-coupling or by cross-metathesis catalysed by first generation Grubbs catalyst, which tolerate phenolic hydroxyl and ketone groups.

The health benefit of fruit juices have been ascribed, in part, to phenolic antioxidants. The antioxidant potential of a range of fruit juices was assessed by measurement of their ability to reduce a synthetic free radical, potassium nitrosodisulphonate, and also by their ability to reduce Fe(III). Vitamin C was found to account for 65–100% of the antioxidant potential of beverages derived from citrus fruit but less than 5% of apple and pineapple juice. The contribution of carotenoids to antioxidant potential was negligible. Although phenolics appear to be major contributors to the antioxidant potential of the non-citrus juices, their identity and bio-availability requires further investigation.

Quercetin is a plant polyphenol which is present in the diet as an aglycone and as sugar conjugates. Despite potent vasodilatory and antioxidant effects in vitro, destruction by intestinal organisms has been assumed to limit its nutritional relevance in the rat. However, we have refined extraction techniques using beta-glucuronidase followed by acid hydrolysis. Following this with HPLC methodology with post-column derivatisation, we have detected significant concentrations of quercetin and its metabolite, isorhamnetin, in tissues of rats maintained on quercetin-rich diets. Percentage recoveries are greater than 95% and intra-batch variation does not exceed 7% suggesting that the method may be useful in further studies of the biological role of this flavonoid.

Effects of three highly saturated fat diets containing either palm fat (RBDPO), hydrogenated soya fat (HSO) or hydrogenated rapeseed fat (HRSO), on short-term indicators of coronary heart disease (CHD) risk in healthy Scottish volunteers were compared. “Free living” subjects (53) were randomly assigned to one of three experimental diets or the “wild” habitual diet group for eight week periods. Fasting blood samples were collected at week 0 (baseline) and at two week intervals. Plasma lipoprotein-cholesterol, triglycerides and indices of lipid peroxidation were assessed. RBDPO diets increased plasma total-, LDL- and HDL-cholesterol (i.e. both beneficial and detrimental effects); HRSO and HSO diets decreased total- and HDL-cholesterol. Changes in cholesterol in ‘wild’ volunteers were similar (0.4m.mol./L) to those in experimental groups (0.4–0.5m.mol./L) with similar absolute values. Plasma triglycerides were reduced after four weeks RBDPO but not HSO or HRSO; values in the “wild” group did not differ from baseline or from other groups. RBDPO reduced indices of lipid peroxidation. HSO increased whole-blood aggregation and plasma conjugated dienes (CD). Physiological relevance of such small changes in indices of CHD risk elicited by the saturated diets are questionable.

The effects of the flavonoids quercetin and myricetin, and the antihepatotoxic agent silymarin, on hydrogen peroxide-mediated DNA damage in human lymphocytes were determined using alkaline single-cell gel electrophoresis (the comet assay). Treatment with hydrogen peroxide increased the levels of DNA strand breaks and oxidised pyrimidine bases in these cells. Quercetin was protective at concentrations above 10 microM and myricetin decreased oxidant-induced DNA strand breakage at concentrations of 100 microM. Cellular metabolism may alter the antioxidant efficacy of the flavonoids. Silymarin had no protective effect at any of the concentrations tested. None of these flavonoids was itself genotoxic. Neither alpha-tocopherol nor beta-carotene decreased hydrogen peroxide-induced DNA breakage. The differences in effectiveness of these dietary compounds against oxidative DNA damage may be explained by differences in their chemical structure or location within the cell.

Many human degenerative diseases involve free radical processes that nutritional antioxidants may ameliorate or prevent, but the optimum intake of such nutrients has yet to be established. Requirement will depend in part on the level of exposure to exogenous and endogenous reactive oxygen species. Smokers incur a sustained degree of oxidant stress from both of these sources, increasing their requirements for vitamins E and C. Male smokers (n = 50) from a Scottish population with habitually low vitamin E and vitamin C intakes consistently had lower plasma ascorbate concentrations (P < 0.02) and greater susceptibility to hydrogen peroxide-stimulated erythrocyte peroxidation in vitro (P < 0.001) than did nonsmokers (n = 50) from the same population. Erythrocyte vitamin E concentrations increased in a dose-dependent manner during 20 wk of supplementation with 70, 140, 560, and 1050 mg D-alpha-tocopherol/d. In smokers each dose was associated with a significant decrease in susceptibility of erythrocytes to peroxidation (P < 0.001). However, red cells of nonsmokers receiving the 1050-mg supplement had an increased susceptibility to peroxidation. Moreover, prolonged supplementation with D-alpha-tocopherol in nonsmokers induced a decline in plasma ascorbate concentration (P < 0.02) in association with an increasing erythrocyte vitamin E uptake (P < 0.001), and in nonsmokers receiving 1050 mg, the susceptibility to peroxidation also increased (P < 0.001). Thus, vitamin E may have prooxidant activity in nonsmokers at high and prolonged intakes.

Objectives of this study were (a) to assess the extent of oxidative stress elicited in human endothelial cells by n-3, n-6 and n-9 mono- and polyunsaturated fatty acids and by interleukin-1 beta and (b) to determine how such stress influenced glutathione peroxidase (GSHPx) activity. Fatty acids were co-incubated with human vascular endothelial cells (HUVEC) for 24 h. Lipid peroxidation, monitored as conjugated diene (CD) formation, increased 3-4-fold with increasing eicosapentaenoic and docosahexaenoic acids: 2-3-fold with linoleic acid; decreased by 50% with arachidonic acid and was unchanged with oleic acid. Changes in glutathione peroxidase activity mirrored conjugated diene formation in the HUVEC incubated with fatty acids. Interleukin-1 beta also increased glutathione peroxidase and conjugated diene formation; the latter increased enzyme activity dose-dependently suggesting a possible role for this oxidation product in the induction of glutathione peroxidase activity. The ability of fish oil fatty acids to induce antioxidant enzymes, particularly those of the glutathione redox system, may be an important mechanism protecting cells and tissues against oxidative and inflammatory cytokine elicited damage.

We previously reported the identification of a new alpha-tocopherol-binding protein (approximately 15 kDa) in the cytosol of rat liver and heart and in rabbit heart (A.K. Dutta-Roy et al., J. Nutr. Biochem. 5, 562-570, 1994). This protein specifically binds alpha-tocopherol and enhances its transfer between separate membranes. In the present paper we have purified and characterized the alpha-tocopherol-binding protein from bovine heart cytosol and compared its various structural and functional properties with the similar size (approximately 15 kDa) cytosolic fatty acid-binding protein of this tissue. alpha-Tocopherol-binding protein was purified to electrophoretic homogeneity from bovine heart cytosol by a procedure involving precipitation with 70% ammonium sulfate, followed sequentially by gel filtration chromatography and chromatofocusing. The purified protein migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 16 kDa. Isoelectric focusing of the purified protein showed that the pI value is around 4.5. The binding of alpha-tocopherol to the purified protein was rapid, reversible, and saturable. The alpha-tocopherol-binding protein did not bind oleate as assessed by direct radiolabeled fatty acid binding and fluorescence enhancement assay. Amino acid analysis showed the presence of a large number of Ala, Gly, Ser, Lys, and Pro residues and a lesser number of aromatic residues in this protein. Anti-bovine heart fatty acid-binding protein antibody did not recognize the alpha-tocopherol-binding protein in the Western blot. The Western blot, ligand affinity, molecular size, and amino acid analysis data suggest that the alpha-tocopherol-binding protein is different from the cytosolic fatty acid-binding protein and that it may be involved in intracellular transport and metabolism of alpha-tocopherol.

Vitamin E (RRR-α-tocopherol) is a lipid-soluble antioxidant that is present in the membranes of intracellular organelles. There it plays an important role in the suppression of free radical-induced lipid peroxidation. There are eight naturally occurring homologues of vitamin E that differ in their structure and in biological activity in vivo and in vitro. Although γ-tocopherol is a more effective free radical scavenger than α-tocopherol in vitro, the reverse is true in vivo, suggesting that the tocopherol distribution systems favor the localization of α-tocopherol at the sites where it is required. Vitamin E is transported in plasma primarily by lipoproteins, but little is known of how it is transported intracellularly. A 30 kDa α-tocopherol-binding protein in the liver cytoplasm may regulate plasma vitamin E concentrations by preferentially incorporating the vitamin E homologue, RRR-α-tocopherol (α-tocopherol), into nascent very low density lipoproteins. However, this α-tocopherol-binding protein is unique to the hepatocyte, whereas α-tocopherol is present in the cells of all major tissues. Moreover α-tocopherol accumulates at those sites within the cell where oxygen radical production is greatest and thus where it is most required; in the membranes of heavy mitochondria, light mitochondria, and endoplasmic reticulum. This raises the question of how the lipid-soluble α-tocopherol is transported intracellularly in different tissues. We have identified a new α-tocopherol-binding protein of molecular mass 14.2 kDa in the cytosol of heart and liver. This protein specifically binds α-tocopherol in preference to the δ- and γ-homologues but does not bind oleate. Studies on immunoreactivity and ligand specificity of the protein suggest that it is not a fatty acid-binding protein. The 14.2 kDa α-tocopherol-binding protein stimulates the transfer of α-tocopherol from liposomes to mitochondria in vitro by 8 to 10 fold. We suggest that this low molecular mass TBP may be responsible for the intracellular transport and distribution of α-tocopherol in the tissues.

Smokers incur a sustained free radical load that may increase their vitamin E requirement. Erythrocytes of male smokers from a Scottish population with a habitually low vitamin E intake were more susceptible to hydrogen peroxide-stimulated peroxidation than were those from nonsmokers (P < 0.001). Plasma concentrations of lipid peroxides, thiobarbituric acid re- active substances, and conjugated dienes were also elevated in smokers compared with nonsmokers (P < 0.05). These indexes of oxidative stress were markedly decreased (P < 0.001) in the smokers and nonsmokers after consumption of 280 mg dl-a to- copherol acetate/d for 10 wk. Platelet numbers in serum of both smokers and nonsmokers were also decreased by vitamin E sup- plementation (P < 0.02). Although the clinical significance of the results is unclear, elevated indexes of lipid peroxidation are associated with the pathogenesis of atherosclerosis, and platelets are involved with fibrinolysis. Therefore, both smokers and non- smokers may benefit from increased vitamin E intakes. Am J Clin Nutr l994;60:383-7.

An alpha-tocopherol-binding protein (TBP) with a molecular mass of 14.2 kDa has been identified from the cytosol of rat heart and liver and purified to electrophoretic homogeneity by precipitation with 70% ammonium sulphate, followed by gel filtration and ion-exchange chromatography. In addition to the 14.2 kDa TBP, liver also contains the previously described 30 kDa TBP. The concentrations of the 14.2 kDa TBP in heart and liver were 12.3 micrograms and 17.5 micrograms per g of tissue, respectively. The purified protein specifically binds d alpha-tocopherol in preference to the delta- and gamma-homologues but does not bind oleate. The TBP stimulated the transfer of d alpha-tocopherol from liposomes to mitochondria in vitro by 8-10 fold. These results suggest that low molecular mass TBPs may play a role in intracellular vitamin E transport.

We have measured coronary heart disease (CHD) risk factors, dietary antioxidant intakes, plasma antioxidant concentrations, activities of blood antioxidant enzymes and plasma indices of lipid peroxidation in current smokers, past-smokers and those who have never smoked (sample size=242; mean age±SE=54±lyr). Compared with non-smokers, both current smokers and past-smokers had increased plasma concentrations of low density lipoproteins (LDL) (P=0.027) and triglycerides (P

Epidemiological studies of cardiovascular disease have resulted in the identification of more than 250 risk factors. However, ascribing causality to a risk factor requires knowledge of the biochemical events involved in the pathogenesis of the disease. The ‘lipid hypothesis’, which relates the risk of developing cardiovascular disease to high saturated fat intake and elevated plasma cholesterol levels, is criticized because most intervention trials have failed to correlate the reduction of plasma cholesterol levels by dietary and pharmacological means with a reduction in the incidence of cardiovascular disease. The ‘antioxidant hypothesis’ develops the lipid hypothesis by suggesting that oxidized cholesterol arising from insufficient antioxidant intake and increased free-radical activity, rather than cholesterol per se, is a risk factor. The convergence of biochemical and epidemiological studies may eventually elucidate the cellular mechanisms responsible for the development of cardiovascular disease.

When stress-susceptible and stress-resistant pigs consumed diets containing 10 iu vitamin E kg-1, the stress-susceptible pigs had damaged cell membranes. This was indicated by increased plasma activities of creatine kinase and pyruvate kinase. Plasma concentrations of thiobarbituric acid reactive substances and conjugated dienes were also increased suggesting that the membrane damage was caused by free radicals. Susceptibility to free radicals was further demonstrated by an increased tendency for erythrocytes and tissue homogenates from stress-susceptible pigs to peroxidise when incubated with hydrogen peroxide and iron, respectively. Supplementation of the diets with approximately 20 times normal requirements of vitamin E decreased plasma creatine kinase and pyruvate kinase activities, and inhibited the formation of the indices of peroxidation in vivo and in vitro. It is concluded that the stress-susceptible pig has an abnormality in its antioxidant defence mechanisms. However, this abnormality was not due to a deficiency in selenium-dependent glutathione peroxidase, since activities were significantly increased in the longissimus dorsi of stress-susceptible pigs. Although the nature of the antioxidant defect is unclear, it is suggested that stress-susceptible pigs are under a sustained oxidant stress and that a decreased ability to accommodate even a normal free radical load may contribute to the rapid development of the fatal stress response.

1. 1. The effects of cannulation of the dorsal aorta of dogfish on selected respiratory and haematological parameters has been assessed over a 24 hr period. 2. 2. Immediately after operational procedures pHaPaO2 and CaO2 were low and CaCO2, and plasma lactate were high. Erythrocyte swelling and a leucopenia were apparent. 3. 3. Cardio-ventilatory coupling was observed in only 2 individuals. 4. 4. The majority of measured variables attained a stable level within 3 hr, exceptions being pH and lactate. However, it is argued that even after 24 hr post-operation the fish were in a “stressed” condition.

Halothane-reactor and non-reactor pigs were offered diets containing either 10 or 135 I.U. vitamin E kg −1 ad libitum for 6 weeks. There were no differences in food intake or weight gain between groups. The fat contents of the semi-tendinosus, gluteus medius and serratus muscles of reactors were significantly less than those of non-reactors irrespective of dietary vitamin E content. Vitamin E supplementation of diets significantly elevated plasma and tissue vitamin E concentrations of both reactors and non-reactors. However, after vitamin E supplementation, significantly higher vitamin E concentrations were found in muscles of reactors compared to non-reactors. A similar trend was apparent when the tissues of non-supplemented reactors and non-reactors were compared. There were no indications of antioxidant enzyme deficiencies in reactors. Plasma pyruvate kinase and creatine kinase activities did not discriminate between reactors and non-reactors. However, after application of a standard pharmacological stress (neostigmine bromide), plasma pyruvate kinase activities of reactors were higher than those of non-reactors irrespective of vitamin E supplementation of the diet. The possibility that vitamin E function is compromised in stress susceptible pigs, and that this may be compensated for by dietary vitamin E supplementation, is discussed.